Uptake of antibiotics by human polymorphonuclear leukocyte cytoplasts.

Enucleated human polymorphonuclear leukocytes (PMN cytoplasts), which have no nuclei and only a few granules, retain many of the functions of intact neutrophils. To better define the mechanisms and intracellular sites of antimicrobial agent accumulation in human neutrophils, we studied the antibiotic uptake process in PMN cytoplasts. Entry of eight radiolabeled antibiotics into PMN cytoplasts was determined by means of a velocity gradient centrifugation technique. Uptakes of these antibiotics by cytoplasts were compared with our findings in intact PMN. Penicillin entered both intact PMN and cytoplasts poorly. Metronidazole achieved a concentration in cytoplasts (and PMN) equal to or somewhat less than the extracellular concentration. Chloramphenicol, a lipid-soluble drug, and trimethoprim were concentrated three- to fourfold by cytoplasts. An unusual finding was that trimethroprim, unlike other tested antibiotics, was accumulated by cytoplasts more readily at 25 degrees C than at 37 degrees C. After an initial rapid association with cytoplasts, cell-associated imipenem declined progressively with time. Clindamycin and two macrolide antibiotics (roxithromycin, erythromycin) were concentrated 7- to 14-fold by cytoplasts. This indicates that cytoplasmic granules are not essential for accumulation of these drugs. Adenosine inhibited cytoplast uptake of clindamycin, which enters intact phagocytic cells by the membrane nucleoside transport system. Roxithromycin uptake by cytoplasts was inhibited by phagocytosis, which may reduce the number of cell membrane sites available for the transport of macrolides. These studies have added to our understanding of uptake mechanisms for antibiotics which are highly concentrated in phagocytes.

Antibiotic inhibition of the respiratory burst response in human polymorphonuclear leukocytes.

Recently we found that certain antibiotics which are markedly concentrated by human polymorphonuclear leukocytes (PMN) failed to kill susceptible, intraphagocytic Staphylococcus aureus, even though cellular drug levels were quite high. The possibility that specific antibiotics might adversely affect phagocyte antibacterial function was considered. Thus, we studied the effects of multiple antibiotics and adenosine, a known modulator of the PMN respiratory burst response, on neutrophil antibacterial function. At nontoxic concentrations, these drugs had no effect on degranulation in stimulated PMN. Adenosine was a potent inhibitor of formyl-methionyl-leucyl-phenylalanine (FMPL)-stimulated superoxide and hydrogen peroxide generation in PMN but produced less inhibition of microbial particle-induced respiratory burst activity. Three of the tested antibiotics, all of which reach high concentrations in phagocytic cells, had a marked modulatory effect on the PMN respiratory burst. Clindamycin, which enters phagocytes by the cell membrane adenosine (nucleoside) transport system, had only a modest effect on FMLP-mediated superoxide production but inhibited the microbial particle-induced response by approximately 50%. Roxithromycin and trimethoprim were efficient inhibitors of PMN superoxide generation stimulated by FMLP and concanavalin A (also inhibited by erythromycin) but had less effect on zymosan-mediated respiratory burst activity. Antibiotics which entered phagocytes less readily had no effect on the respiratory burst response in PMN. These results, as well as those of experiments with inhibitors of cell membrane nucleoside receptors, indicated that the antibiotic effect is mediated through intraphagocytic pathways. The possibility that antibiotic-associated inhibition of the PMN respiratory burst response might alter leukocyte antimicrobial and inflammatory function deserves further evaluation.

Pentoxifylline modulation of plasma membrane functions in human polymorphonuclear leukocytes.

Pentoxifylline is known to have major effects on cell membrane function in mammalian cells, including human leukocytes. The protective effects of this agent in animal models of infection and inflammation may be due to alterations in phagocyte (neutrophil and macrophage) function. However, the exact mechanism of action of pentoxifylline is unknown. In this study, we evaluated the effect of the drug on several membrane-associated activities in human polymorphonuclear neutrophils and investigated possible mechanisms for the observed changes in neutrophil function. Pentoxifylline inhibited ingestion of microbial particles (Staphylococcus aureus and zymosan); decreased superoxide generation activated by zymosan, formyl-methionyl-leucyl-phenylalanine, and concanavalin A (but not phorbol myristate acetate); and decreased uptake (transport) of adenosine stimulated by formyl-methionyl-leucyl-phenylalanine and zymosan. In contrast, pentoxifylline actually increased clindamycin uptake in zymosan-stimulated polymorphonuclear neutrophils. However, pentoxifylline had no effect on uptake of adenosine or clindamycin in unstimulated neutrophils. In comparison with known inhibitors of nucleoside transport (nitrobenzylthioinosine and dipyridamole), the results suggested that pentoxifylline does not bind to membrane nucleoside transport receptors. At concentrations which inhibit neutrophil function, pentoxifylline activity is not mediated through external membrane nucleoside regulatory sites. Thus, pentoxifylline affects the activation signal chain at a point beyond the membrane receptors. Whatever its precise mechanism of action, pentoxifylline has a striking modulatory effect on cell membrane-associated responses in stimulated leukocytes and may prove useful for control of injurious inflammatory states.

The entry of antibiotics into human monocytes.

Effective therapy of infections due to facultative intracellular micro-organisms, which persist after ingestion by mononuclear phagocytic cells, requires the use of antibiotics with the ability to inactivate these intra-phagocytic bacteria. Since entry of antibiotics into mononuclear phagocytes is a pre-requisite for activity against such intracellular organisms, we have determined the uptake of 11 radiolabelled antibiotics by human peripheral blood monocytes. beta-Lactams (penicillin G, cefamandole and cefotaxime), gentamicin, and metronidazole had a limited ability to enter monocytes, achieving cellular concentrations which were equal to or less than extracellular levels (C/E less than or equal to 1). Imipenem, a novel beta-lactam antibiotic, rapidly bound to monocytes, but cell-associated drug progressively declined during further incubation. Chloramphenicol, a lipid-soluble drug, and trimethoprim were concentrated three-fold by human monocytes. In comparison with the other antibiotics, roxithromycin (C/E = 14), clindamycin (C/E = 6 to 7) and erythromycin propionate (C/E = 4 to 5) were markedly concentrated by human monocytes. In contrast to our findings in other phagocytes, there was no evidence that active membrane transport was involved in monocyte uptake of clindamycin, erythromycin and roxithromycin. We have demonstrated that several antibiotics are highly concentrated within human monocytes, and it will be important to evaluate the effects of this antibiotic uptake on various monocyte functions, including intra-phagocytic antibacterial activity.

Contrasts between phagocyte antibiotic uptake and subsequent intracellular bactericidal activity.

An ideal antibiotic for therapy of infections due to facultative intracellular organisms would enter phagocytes readily and kill intracellular bacteria. We have examined the consequences of antibiotic uptake by human polymorphonuclear lymphocytes (PMN) on intraphagocytic bactericidal activity, using antibiotics which differ markedly in their ability to enter PMN. After ingestion of Staphylococcus aureus, PMN were evaluated in regard to uptake of antibiotics and survival of intraphagocytic bacteria in the presence or absence of these drugs. Except for erythromycin, the uptake of which was slightly decreased, the entry of tested antibiotics into PMN was increased or unchanged after ingestion of S. aureus. Clindamycin and erythromycin, which achieved high cellular levels in PMN, failed to produce a significant reduction in viable intraphagocytic S. aureus during 3 h of antibiotic exposure. In contrast, rifampin, which was concentrated severalfold by phagocytes, was able to kill intracellular staphylococci. Gentamicin and penicillin G penetrated PMN rather poorly. However, while gentamicin demonstrated efficient intraphagocytic killing of bacteria, penicillin had no intracellular effect during the 3-h incubation period. These observations document that the ability to enter phagocytes is only one of the factors which determine the intracellular antibacterial activity of an antibiotic.

Antibiotic uptake by alveolar macrophages of smokers.

Cigarette smoking, particularly when associated with chronic pulmonary disease, increases the risk of respiratory tract infection. Thus, we elevated the uptake of antibiotics by alveolar macrophages (AM) obtained by bronchoalveolar lavage from persons who smoke and have associated pulmonary abnormalities, circumstances which adversely affect certain macrophage functions. The entry of radiolabeled drugs into AM was determined by a velocity-gradient centrifugation technique, and uptake was expressed as the ratio of cellular to extracellular antibiotic concentration (C/E). Cefamandole and penicillin G were taken up poorly by the AM obtained from smokers (C/E less than or equal to 1). Cellular levels of isoniazid, gentamicin, and tetracycline were similar to their extracellular concentrations. The lipid-soluble drugs lincomycin, chloramphenicol, and rifampin were concentrated severalfold by the AM from smokers (C/E = 3 to 11). Ethambutol also entered macrophages readily (C/E = 11). Erythromycin and clindamycin were massively concentrated by the AM from smokers (C/E = 23 to 56). The AM of smokers accumulated a lipid-soluble antibiotic (rifampin) and actively transported agents (erythromycin propionate, clindamycin) more avidly than did the AM of nonsmokers. Augmented uptake of these antibiotics by the AM of smokers may be related to structural and functional alterations induced by smoking.

Influence of bacterial-antibiotic interactions on subsequent antimicrobial activity of alveolar macrophages.

The efficacy of an antibiotic in the treatment of bacterial infections depends upon the interactions of drug, bacteria, and phagocytes. In an examination of certain of these interactions, overnight cultures of 3H-labeled Staphylococcus aureus were briefly incubated in the presence or absence of various antibiotics prior to phagocytosis by rabbit alveolar macrophages. Preincubation of organisms with an inhibitor of bacterial protein synthesis (clindamycin, erythromycin, chloramphenicol, or rifampin) but not with a cell wall-active drug (penicillin G or cefazolin) led to an increase in phagocytosis and early intracellular killing by alveolar macrophages. Antibiotic-mediated susceptibility of S aureus to ingestion by alveolar macrophages correlated with the inhibition of bacterial protein synthesis. Clindamycin-treated staphylococci were more efficiently opsonized by heat-inactivated serum and bound larger amounts of specific antibody than did control organisms. Thus, compromise of one or more antiphagocytic surface components secondary to decreased protein synthesis is the means by which certain antibiotics influence bacterial susceptibility to the antimicrobial mechanisms of phagocytes. This effect may be an important determinant in the success of treatment with inhibitors of bacterial protein synthesis.

Interactions of antibiotics and phagocytes.

Optimal therapy of infections due to organisms capable of surviving within phagocytes would include use of antimicrobials that penetrate phagocytic cells and inactivate intracellular organisms. To establish those characteristics of drug and cell that mediate the antibiotic-phagocyte interaction, we have studied the uptake of radiolabelled antibiotics by rabbit alveolar macrophages (AM), human AM from smokers and non-smokers, and human polymorphonuclear leukocytes (PMN). Relative entries of drug groups into the three types of phagocytic cells were similar. Penicillin G and cephalosporin antibiotics were taken up poorly by phagocytes. Lipid-soluble antibiotics, such as rifampicin and chloramphenicol, were concentrated several-fold (2-5) by phagocytes. Ethambutol, erythromycin and clindamycin were concentrated many-fold (5-50) by phagocytic cells. Human AM of smokers accumulated certain antibiotics more avidly than AM of non-smokers. Clindamycin entry into phagocytes was shown to be an active, energy-requiring process, mediated by the nucleoside transport system. Ingestion of microbial particles by PMN stimulated transport of both clindamycin and nucleoside (adenosine) into the cell.

Effect of erythrocyte ingestion on macrophage antibacterial function.

Individuals with sickle cell anemia are subject to serious infections caused by a number of bacteria, including Salmonella species and Staphylococcus aureus. It has been suggested that in sickle cell anemia, extensive erythrophagocytosis by macrophages may interfere with their antibacterial function and thereby predispose to infection. As a means of investigating this possibility, we evaluated the effects of erythrocyte ingestion on the Killing of Salmonella typhimurium by peritoneal macrophages and of S. aureus by alveolar macrophages. Monolayers of rabbit macrophages were exposed to erythrocytes or latex particles immediately before and during bacterial challenge. Erythrophagocytosis markedly inhibited intracellular killing of S. typhimurium by peritoneal macrophages (bacterial survival was 181% of control) and of staphylococci by alveolar macrophages (bacterial survival was greater than 200% of control). Exposure to latex particles depressed the bactericidal activity of alveolar macrophages to a lesser degree. Next we investigated the possibility that erythrophagocytosis inhibits oxidative bactericidal mechanisms in macrophages. Hexose monophosphate shunt activity was stimulated by erythrocyte ingestion. However, zymosan-induced superoxide generation and chemiluminescence were suppressed by erythrocytes. Furthermore, a cell-free (hypoxanthine-xanthine oxidase) system for chemiluminescence generation was also depressed in the presence of erythrocytes (intact or lysate) or by purified hemoglobin. These studies reveal that erythrophagocytosis inhibits macrophage antibacterial function, probably because of interactions between erythrocyte components and reactive products of phagocyte oxygen metabolism. This host defense abnormality may predispose to bacterial infection in certain hemolytic anemias.

Membrane transport of clindamycin in alveolar macrophages.

The use of antibiotics which can penetrate phagocytic cells and kill intracellular organisms is desirable in the treatment of chronic facultative bacterial infections. Recently, we reported that several antibiotics were selectively concentrated by rabbit alveolar macrophages. Clindamycin accumulation was especially marked. In the present study we evaluated the plasma membrane transport (initial uptake) of clindamycin in alveolar macrophages. The transport of clindamycin is an active process, as documented by requirements for cellular viability, elevated environmental temperature, metabolic energy, and establishment of the 40- to 50-fold cellular/extracellular gradient. Energy for membrane transport of the drug depended at least in part upon mitochondrial oxidative respiration and cell membrane Na-K pump activity. Kinetic analysis of active clindamycin transport revealed it to be saturable, with a high binding affinity (Km = 1 mM) and a high velocity of uptake (Vmax = 15.8 nmol/45 s per 10(6) cells). Clindamycin uptake was not influenced by the presence of hexose or amino acids, but was inhibited by nucleosides (adenosine, puromycin). Decreased clindamycin transport in the presence of puromycin was typical of competitive inhibition (increased Km, unchanged Vmax). Conversely, competitive inhibition of adenosine transport by clindamycin was documented. Thus, clindamycin is transported into alveolar macrophages via the nucleoside system. The potential biological consequences of this unique antibiotic transport mechanism are of interest.